Musa M. Mhlanga

Visualizing the distribution and transport of mRNAs in living cells

We have visualized the movements of native mRNAs in living cells. Using nuclease-resistant molecular beacons, we imaged the transport and localization of oskar mRNA in Drosophila melanogaster oocytes. When the localization pattern was altered by genetic manipulation of the mRNAs 3′ untranslated region, or by chemical perturbation of the intracellular tubulin network, the distribution of the fluorescence signals changed accordingly. We tracked the migration of oskar mRNA in real time, from the nurse cells where it is produced to the posterior cortex of the oocyte where it is localized. Our observations reveal the presence of a transient, and heretofore elusive, stage in the transport of oskar mRNA. Direct visualization of specific mRNAs in living cells with molecular beacons will accelerate studies of intracellular RNA trafficking and localization, just as the use of green fluorescent protein has stimulated the study of specific proteins in vivo.

tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells

When oligonucleotide probes are microinjected into cells to image the distribution of RNAs, they are rapidly sequestered into the nucleus. As a result, it is difficult to detect mRNAs in the cytoplasm of living cells. We were able to overcome this process by attaching tRNA transcripts to the probes. We show that when fluorescently labeled tRNAs, tRNAs with extensions at their 5′ end, or chimeric molecules in which a molecular beacon possessing a 2′-O-methylribonucleotide backbone is linked to a tRNA, are injected into the nucleus of HeLa cells, they are exported into the cytoplasm. When these constructs are introduced into the cytoplasm, they remain cytoplasmic. These constructs allow the distribution of both the general mRNA population and specific mRNAs to be imaged in living cells. This strategy should also be useful for enhancing the efficacy of antisense oligonucleotides by keeping them in the cytoplasm. Our observations show that the fidelity of the tRNA export system is relaxed for unnatural tRNA variants when they are introduced into the nucleus in large amounts.

In vivo colocalisation of oskar mRNA and trans-acting proteins revealed by quantitative imaging of the Drosophila oocyte.

Efficient mRNA transport in eukaryotes requires highly orchestrated relationships between nuclear and cytoplasmic proteins. For oskar mRNA, the Drosophila posterior determinant, these spatio-temporal requirements remain opaque during its multi-step transport process. By in vivo covisualization of oskar mRNA with Staufen, its putative trafficking protein, we find oskar mRNA to be present in particles distinct from Staufen for part of its transport. oskar mRNA stably associated with Staufen near the posterior pole. We observe oskar mRNA to oligomerize as hundreds of copies forming large particles which are necessary for its long range transport and localization. We show the formation of these particles occurs in the nurse cell nucleus in an Hrp48-dependent manner. We present a more refined model of oskar mRNA transport in the Drosophila oocyte.

Using tRNA-linked molecular beacons to image cytoplasmic mRNAs in live cells

Imaging products of gene expression in live cells will provide unique insights into the biology of cells. Molecular beacons make attractive probes for imaging mRNA in live cells as they can report the presence of an RNA target by turning on the fluorescence of a quenched fluorophore. However, when oligonucleotide probes are introduced into cells, they are rapidly sequestered in the nucleus, making the detection of cytoplasmic mRNAs difficult. We have shown that if a molecular beacon is linked to a tRNA, it stays in the cytoplasm and permits detection of cytoplasmic mRNAs. Here we describe two methods of linking molecular beacons to tRNA and show how the joint molecules can be used for imaging an mRNA that is normally present in the cytoplasm in live cultured cells. This protocol should take a total of 4 d to complete.

A 12p array to identify amplified and overexpressed sequences in testicular germ cell tumors

Testicular germ cell tumors (TGCTs) show, for the most part, a characteristic cytogenetic aberration carrying one or more copies of i(12p). Mostly i(12p)-negative TGCTs also show an overrepresentation of 12p11p12 sequences. Critical genes in the amplicon defined by 12p11p12 may be involved in the progression of TGCTs and other tumors. To identify directly the expressed sequence tags (ESTs) involved in the amplicon, we performed comparative genomic hybridization array analysis of five TGCTs demonstrated to exhibit high DNA copy number in this region. The use of arrays spotted with 8,060 IMAGE complementary DNA clones amplified by means of the polymerase chain reaction (PCR) identified 18 amplified ESTs. Most of them were mapped to 12p and the remainder have been mapped to 12. We also identified new testis-associated transcripts by PCR with reverse transcription of the amplified EST sequences. Using combined RNA-array and real-time-beacon PCR analysis, we identified two amplified and overexpressed ESTs. The level of expression in these ESTs was four- to sixfold higher in one tumor sample compared with normal testis. The expression ratios obtained by real-time-beacon PCR analysis correlated with those obtained by RNA-array data analysis, thus validating the techniques used. However, we failed to find an amplified and overexpressed EST commonly overexpressed in all the tumor samples tested, perhaps because the arrays used might only represent approximately 10% of human genes. Therefore we decided to design a 12p array containing 768 ESTs, representing all the 12p-mapped ESTs found in the available databases. Results of comparative genomic hybridization and RNA-array analysis of TGCTs using the 12p array will be presented.